258 



THE DIPHTHERIA BACILLUS 



rotten and from which the sugars had vanished. Park and Williams in their 

 investigations used a broth previously made alkaline with soda : Mace 

 employed a broth to which calcium carbonate had been added. None of 

 these methods however give results as good as those obtainable by the method 

 devised by Martin. This observer succeeded in finding a medium in which 

 no acid reaction is developed and which yields highly toxic cultures. 



1. Method of Roux and Martin. The bacillus is grown in a current of air, 

 and for this purpose a flask (modified from Fernbach's) similar to that shown 

 in the illustration (fig. 170) is very convenient. 



FIG. 170. Flask arranged for the growth of the diphtheria bacillus for toxin 

 production (Roux and Martin's method). 



Pour into each flask 400-500 c.c. of veal broth ; this quantity should not form 

 a layer more than 2 or 3 cm. deep, and the surface of the broth should be below the 

 opening of the lateral tube D. Plug the lateral tube D and the neck of the flask C 

 with cotton- wool : autoclave the flask and its contents : allow to cool and sow the 

 medium through the neck C. Incubate at 37 C. and after about 24 hours or so 

 when the growth is well started and the broth has become cloudy arrange the flask 

 so that a current of air can be passed over the surface of the broth, thus : Into the 

 neck of the flask B and above the wool plug fit an india-rubber plug through which 

 a piece of glass tubing, 6, bent at right angles is passed, and connect this with a 

 second flask A containing a little water through which the air is made to bubble. 

 Attach the tube D by means of a piece of india-rubber tubing to a water pump. 

 When the water is turned on air is bubbled through the water in A where it is satu- 

 rated with moisture and drawn over the surface of the culture in B which is thus 

 aerated. By means of a clip on the tube connecting the flasks A and B the amount 

 of air can easily be regulated so that a constant 'but not violent stream of air can be 

 drawn through the flask. 



After incubating for 3 or 4 weeks the culture is sufficiently rich in toxin. 

 At the bottom of the flask there is a deposit of micro-organisms and on the 

 surface a thin layer of young bacilli. The reaction is strongly alkaline. 



The culture is now filtered through a Chamberland bougie by one of the 

 methods already described (Chap. I.). The filtrate kills adult guinea-pigs 

 in doses of O'l c.c. 



2. Martin's method. Method recommended. The need for the current 

 of air is obviated, and hence also the necessity for complicated apparatus : 

 it is quicker than the method just described and yields moreover a more 

 powerful toxin. A dose of -* J^th c.c. of this toxin is sufficient to kill a guinea- 



Pig- 



Martin uses a peptonized veal broth (p. 33) sterilized by filtration 1 and 



1 If sterilized at 120 C. this medium does not give such good results. It is better to 

 filter or, failing nitration, to sterilize on three successive days at 100 C. 



